Amalgamating tools of molecular biology, genetics, biochemistry, and cell biology, this collaborative competing continuation application offers an interdisciplinary dissection of the nucleoside and nucleobase transporters from Leishmania. Because protozoan parasites are incapable of synthesizing purine nucleotides or nucleobases de novo, purine transporters provide an important, if not obligatory, nutritional function for the parasite and suggest several therapeutic paradigms. In the course of the previously funded investigations, we have cloned and functionally characterized two equilibrative nucleoside transporter (ENT) genes from L. donovani; LdNT1, the gene encoding the adenosine-pyrimidine nucleoside transporter, and LdNT2, the inosine-guanosine transporter gene. We have also created Aldntl and Aldnt2 knockouts by targeted gene replacement and characterized mutationally derived parasites deficient in either LdNT1 or LdNT2 activity. More recently, two additional ENT family members have been identified within the Leishmania major genome project database; LmaNT3, which recognizes purine nucleobases but not nucleosides, and a previously unidentified open reading frame, LmaNT4, which has recently been shown to possess nucleobase transport activity. These molecular and cellular reagents are the cornerstone of the three Specific Aims of this proposal. The first Specific Aim will examine the functional roles performed by these transporters in intact L. major parasites using targeted gene replacement strategies. We will test LmaNT1, LmaNT2, LmaNT3, and LmaNT4 function in L. major promastigotes, metacyclics, and infectious amastigotes by creating and characterizing deltalmant1, deltamant2, deltamant3, and deltalmant4 knockouts in various permutations. In Specific Aim II, we will implement an unbiased genetic screen for Imant2 loss-of-function mutants. This will enable us to identify in a nonintuitive manner key residues within LmaNT2 that are required for either permeation or ligand recognition. The final Specific Aim will be to functionally characterize the novel LmaNT4 nucleobase transporter in Xenopus laevis oocytes. We will determine the ligand specificity and affinities for LmaNT4 and evaluate LmaNT4 expression throughout the L. major life cycle.